T. gondii is an obligate intracellular category B priority pathogen that causes the disease toxoplasmosis and is a model system for other priority B protozoa including Cryptosporidium parvum and Cyclospora caytanensis. Our long-term objective of this grant application is to understand molecular mechanisms used by T. gondii and related protozoa to evade the innate immune response during infection. These studies may also identify novel intracellular mechanisms used by innate immune cells to control intracellular pathogens. As a tool for these studies, we isolated a panel of T. gondii insertional mutants that replicate normally in naive macrophages but have a marked increase in their susceptibility to macrophage activation. In preliminary studies, the majority of these mutants also appear defective for pathogenesis in vivo. Our hypothesis is that these parasite genes interact directly or indirectly with host proteins to modulate intracellular signaling cascades in innate immune cells or enhance resistance to host intracellular mediators. Identification of parasite genes that counter the host innate immune response will facilitate dissection of molecular mechanisms involved in pathogen adaptation to physiological stresses, mechanisms of immune evasion, and intracellular trafficking of parasite products. The specific aims are to: 1. Characterize the T. gondii genes important for evasion of intracellular killing by macrophages (counter-immune genes {cim}) by isolating the parasite genes and using the identified cim genes to complement the mutant strains. 2. Evaluate the function of the cim proteins by determining their localization in parasites and infected macrophages and analyzing their role in intracellular survival in activated macrophages using a variety of molecular, cellular and immunologic techniques. 3. Analyze the role of the cim proteins in T. gondii survival and dissemination using a murine model of pathogenesis. As part of this aim, we will test the hypothesis that cim proteins enhance parasite resistance to IFN-gamma-dependent immune mechanisms in vivo. The studies proposed here offer important insight into the means by which Apicomplexan parasites establish an environment for their survival in innate immune cells in the midst of an inflammatory microenvironment thus contributing to our understanding of the molecular basis of parasite pathogenesis that is relevant to human toxoplasmosis.